Orientador: Carla Beatriz Grespan Bottoli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T16:02:57Z (GMT). No. of bitstreams: 1 Andre_MarceloFabiano_D.pdf: 2661780 bytes, checksum: 3f8c06d00ddf653d50feed242ea04514 (MD5) Previous issue date: 2014 / Resumo: Antioxidantes naturais são metabólitos secundários presentes em uma gama variada de plantas medicinais com propriedades específicas. Podem apresentar ação tranqüilizante, analgésica, antiinflamatória, citotóxica, anticoncepcional, antimicrobiana, antiviral e fungicida.Vários metabólitos secundários são conhecidos. Os três principais grupos são os terpenos, os polifenóis (flavonóides) e os alcalóides. A carqueja foi escolhida por apresentar em sua composição vários compostos fenólicos (antioxidantes), tais como a quercetrina, a rutina e o canferol. Estes flavonóides de origem natural são fisiologicamente ativos, isto é, apresentam propriedades anti-inflamatórias, antifragilidade capilar e antitumoral, respectivamente, além de serem antioxidantes. O trabalho foi realizado em duas etapas, a saber, a primeira etapa foi realizada no Brasil, e teve como objetivo, o desenvolvimento e validação de métodos para determinação de compostos fenólicos (ácidos fenólicos e flavonóides) em extrato de carqueja empregando técnicas cromatográficas e eletroforéticas e análise de algumas amostras comerciais de diferentes regiões. A segunda etapa foi realizada na Espanha, tendo como objetivos o desenvolvimento e validação do método empregando eletroforese capilar acoplada a um espectrômetro de massas (CE-ESI-TOF), utilizando a técnica de extração com líquido pressurizado (PLE), considerada avançada e verde. Também na Espanha, foi realizada a determinação de fenóis totais e atividade antioxidante empregando os ensaios de Folin e DPPH, respectivamente. O desenvolvimento e preparo de amostra incluiu a otimização das etapas de extração e hidrólise dos compostos fenólicos presentes na carqueja. A quantificação pelas técnicas cromatográficas foi realizada empregando coluna com fase estacionária C-18 e eluição por gradiente, para as técnicas eletroforéticas foi utilizado meio tamponante (tetraborato de potássio, pH 10) com 10% de modificador orgânico. Os métodos foram validados segundo orientações da ANVISA. Foram analisadas amostras comerciais de três estados, a saber, São Paulo, Minas Gerais e Bahia. A amostra do Estado da Bahia apresentou o maior teor de concetração dos compostos fenólicos estudados, excetuando o ácido gálico que está em maior teor de concentração na amostra do Estado de Minas Gerais. Comparando a concentração destes compostos nas diferentes amostras, através da determinação do teor por UHPLC-MS/MS e HPLC-UV, ao nível de 5% de significância, não houve diferença significativa entre as técnicas / Abstract: Natural antioxidants are secondary metabolites in a wide range of medicinal plants with specific properties. May present action tranquilizer, analgesic, anti-inflammatory, cytotoxic, contraceptive, antimicrobial, antiviral and fungicida.Vários secondary metabolites are known. The three main groups are terpenes, polyphenols (flavonoids) and alkaloids. Carqueja was chosen because it has in its composition various phenolic compounds (antioxidants), such as quercetrin, rutin and kaempferol. These flavonoids are naturally occurring physiologically active, i.e., exhibit anti-inflammatory, antitumor and antifragilidade capillary, respectively, and are antioxidants. The work was carried out in two stages, namely, the first step was made in Brazil, and aimed to the development and validation of methods for the determination of phenolic compounds (phenolic acids and flavonoids) in gorse extract using chromatographic and electrophoretic techniques and analysis of some commercial samples of different regions. The second stage was conducted in Spain, having as objective the development and validation of the method using capillary electrophoresis coupled to mass spectrometry (CE-ESI-TOF) using the technique of pressurized liquid extraction (PLE), considered advanced and green . The determination of total phenols and antioxidant activity assays employing Folin and DPPH, respectively was also in Spain held. The development and sample preparation steps included the optimization of the extraction and hydrolysis of phenolic compounds present in the gorse. Quantification was performed by chromatographic techniques employing column with stationary phase C-18 and gradient elution, electrophoretic techniques for buffering medium was used (potassium tetraborate, pH 10) with 10% organic modifier. The methods were validated according to guidelines of ANVISA. Commercial samples were analyzed three states, namely São Paulo, Minas Gerais and Bahia. The sample of Bahia presented the highest level of averaged concentration of phenolic compounds studied, except gallic acid content is greater concentration in the sample of the state of Minas Gerais. Comparing the concentration of these compounds in different samples by determining the content by UHPLC-MS / MS and HPLC-UV at the 5% level of significance, there was no significant difference between the techniques / Doutorado / Quimica Analitica / Doutor em Ciências

De nombreuses études dans la seconde moitié du 20ème siècle, ont mis en évidence que des génotoxiques cancérogènes réagissent avec l'ADN pour former par liaison covalente des adduits qui sont impliqués dans le processus cancérigène. Bien qu’il existe des preuves convaincantes de la présence de multiples adduits à l'ADN dans les poumons de sujets exposés au tabagisme ou en milieu professionnel à un aldéhyde donné, il est évident que c'est un domaine dans lequel des recherches supplémentaires ont été nécessaires. L’objectif de ce travail de thèse est d’établir des profils d’adduits exocycliques à l'ADN induits par le mélange d’aldéhydes, qui pourraient à terme être considérés comme un marqueur génotoxique de l’exposition aux aldéhydes, tant endogène qu’environnemental. Pour cette raison, nous avons validé une méthode en UHPLC-MS/MS rapide, sensible et précise en utilisant la dilution isotopique, pour la quantification à l’état de trace de 9 adduits exocycliques à l’ADN dérivés de 8 principaux aldéhydes exogènes et endogènes, notamment le formaldéhyde, l’acétaldéhyde, l’acroléine, le crotonaldéhyde, le malondialdéhyde, le 4-hydroxy-2-nonénal, le glyoxal et le méthylglyoxal. Ces adduits ont été synthétisés et purifiés ainsi que leurs homologues marqués au 13C10, 15N5, identifiés et quantifiés par le biais des courbes d'étalonnage allant de 0,25 à 250 ng/mL d'adduits dans l'eau et l'ADN afin de décrire les effets matrice. Des échantillons de contrôle qualité ont été préparés et analysés afin de vérifier l'exactitude et la précision de la méthode dans des situations de répétabilité et de fidélité intermédiaire. L'absence de contamination croisée a également été démontrée. La méthode est capable de différencier les 9 analytes d'intérêt et leurs étalons internes en utilisant pour chaque analyte une transition de quantification et une seconde de confirmation. Cette méthode a été validée selon les recommandations de l'Agence Européenne des Médicaments concernant les méthodes bioanalytiques. Elle répond à tous les critères essentiels pour garantir l'acceptabilité des performances et la fiabilité des résultats d'analyse. Cette méthode est la toute première validée et peut être utilisée en adductomique dans le cadre d'études sur l'exposome. En plus, nous avons simultanément mesuré par une approche in vitro les 9 adduits exocycliques dans de l’ADN de thymus de veau exposé à de différentes concentrations de chaque aldéhyde seul ou en mélanges équimolaires. Cette approche nous a permis d’établir des relations dose-dépendantes pour tous les aldéhydes à l’exception du malondialdéhyde et du méthylglyoxal. Une relation dose-réponse a également été observée avec les mélanges équimolaires d’aldéhydes. Elle a permis de définir des réactivités différentes des aldéhydes en mélange vis-à-vis de l’ADN. Les profils de ces adduits exocycliques ont été également déterminés dans l'ADN de sang de fumeurs et de non-fumeurs. La fumée de cigarette contient plusieurs aldéhydes connus de se lier par covalence aux bases de l’ADN, ainsi l’adduit à l’ADN peut être considéré comme biomarqueur d’exposition au tabac. Des différences significatives dans les niveaux d’adduits ont été obtenues entre l’ADN des fumeurs et celui des non-fumeurs à l’exception de l’adduit induit par le malondialdéhyde. Des corrélations ont été établies entre chaque adduit et les marqueurs de la consommation tabagique sans aucune corrélation significative de la totalité des adduits avec un marqueur spécifique. Par ailleurs, nous avons montré que l’exposition au formaldéhyde, au butanal et au benzaldéhyde a eu un effet sur les concentrations du MDA urinaire mesurées chez les policiers libanais stationnés au carrefour pendant 7 h par jour et après exposition de 5 jours aux émissions du trafic routier. Une augmentation du MDA plasmatique a été décrite ; les années de travail avaient une incidence sur les concentrations de ce biomarqueur. / Many studies in the second half of the 20th century have shown that genotoxic carcinogens, either directly or after metabolic activation, react with DNA to form covalently bonded adducts that are absolutely central in the carcinogenic process. Although there is compelling evidence of the presence of multiple DNA adducts in the lungs of subjects exposed to smoking or occupational exposure to a given aldehyde, it is clear that this is an area in which further research has been necessary. The aim of this thesis is to establish exocyclic DNA adducts profiles induced by the mixture of aldehydes, which could eventually be considered as a genotoxic marker of aldehyde exposure, both endogenous environmental. For this reason, we have validated a fast, sensitive and precise method on liquid chromatography coupled to mass spectrometry in tandem mode (UHPLC-MS/MS) using isotopic dilution, for trace quantification of 9 exocyclic DNA adducts derived from 8 major exogenous and endogenous aldehydes, including formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. These adducts were synthesized and purified as well as their labeled homologues, identified and quantified through standard curves ranging from 0.25 (LLOQ) to 250 ng/mL (ULOQ) adducts in water and in DNA to describe the matrix effects. Quality control (QC) samples were prepared and analyzed to verify the accuracy and precision of the method in repeatability and intermediate fidelity situations. The absence of cross-contamination has also been demonstrated. The method is able to differentiate the 9 analytes of interest and their internal standards using for each analyte a quantification transition and a confirmation transition. This method has been validated according to the recommendations of the European Medicines Agency (EMA) concerning bioanalytical methods. It meets all the essential criteria to guarantee the acceptability of the performances and the reliability of the analysis results. This method is the very first validated and can be used in adductomics in the context of studies on the exposome. In addition, the exocyclic adducts were simultaneously measured by an in vitro approach in calf thymus DNA exposed to different concentrations of each aldehyde apart or in equimolar mixtures. This approach allowed us to establish dose-dependent relationships for all aldehydes with the exception of malondialdehyde and methylglyoxal. A dose-response relationship was also observed with equimolar mixtures of aldehydes. It made it possible to define different reactivities of aldehydes in mixture versus DNA. The profiles of these exocyclic adducts were also determined in the blood DNA of smokers and non-smokers. Cigarette smoke contains several aldehydes known to covalently bind to DNA bases, so the DNA adduct may be considered as biomarker of tobacco exposure. Significant differences in adducts levels were obtained between smokers and non-smokers DNA with the exception of malondialdehyde-induced DNA adduct. Correlations were established between each adduct and smoking-related markers without any significant correlation of all adducts with a specific marker. Furthermore, we have shown that exposure to formaldehyde, butanal and benzaldehyde had an effect on the concentrations of urinary MDA measured in Lebanese police stationed at the intersection for 7 hours a day and after 5-day exposure to road traffic. An increase in plasma MDA has been described; years of work had an impact on the concentrations of this biomarker. These results are promising and it would be interesting to validate in population the profile of 9 exocyclic adducts as biomarkers of exposure to both exogenous and endogenous aldehydes as part of an adductomic approach to understand the carcinogenic risk in relation to aldehydes exposures in urban areas.

The aim of the thesis was to analyze the historical pharmaceutical preparations, including the determination of the active substance and identify theirs possible degradation products. A historical pharmaceutical preparation of naloxone was analyzed by mass spectrometry. Historical pharmaceutical preparations of adrenaline and ephedrine were analyzed by UHPLC-MS and were quantified using a calibration curve. In the historical injection solution of naloxone, "NARCAN", dated around 1980, there were no significant degradation products and the measured mass and UV spectrum was consistent with the spectrum of naloxone. The analyzed sample of naloxone was stable even after 35 years of storage. In the analyzed historical injection solution of adrenaline, "Adrenalin Hydrochlor., Dr. Heisler" (dated between 1917 and 1938) was determined 5.26 ± 0.11 % of the declared amount of adrenaline. In the measured spectras were noticeable degradation products, which have not been described in the literature yet and their identification was beyond the scope of this paper. The analyzed sample of adrenaline was almost completely degraded during about ninety years. The stability test carried out with four standard solutions of adrenaline proved influence of oxygen, light, temperature and time on the degradation of adrenaline. In...

Specialized microbial metabolites are described as low-molecular-weight bioactive compounds, which are dispensable for the growth, evolution, or reproduction of its producer. This group of substances includes the lincosamides, which are produced mainly by the bacteria of the Streptomyces genera. Apart from other precursors, two low-molecular-weight thiols, ergothioneine and mycothiol, are essential participants of the lincosamide biosynthesis. Mycothiol (MSH) serves in this pathway as a source of sulphur, on the other hand, ergothioneine (ESH) constitutes a conjugate with the aminosugar moiety of lincosamide structure. The conjugate is condensed with an activated amino acid, which is catalyzed by an unusual enzyme to form a core of the lincosamide molecule. The objective of this diploma thesis is to isolate the conjugate of ESH and aminooctose, which serves as a substrate of the LmbD biosynthetic protein. Another aim is to study the links between the thiol metabolism and the biosynthesis of three lincosamides, lincomycin, celesticetin, and intervencin, which are produced by different bacterial strains. Bacterial strains were cultivated under laboratory conditions and methods of liquid chromatography with UV and MS detection were used for the analysis. The parameters of the methods were developed...

Occurrence, accumulation and subsequent fate of pharmaceuticals in environment currently represent a very actual topic. Worldwide, thousands of tons of pharmaceutical substances are consumed every year. A large portion of pharmaceuticals is, in unchanged or metabolized forms, disposed via sewage systems and wastewater treatment plants. Considering the fact that wastewater treatment processes are not able to completely eliminate all active substances or their metabolites, pharmaceuticals are systematically washing out into the water system and increasingly contaminate the ground and surface waters. The problematics of continuous control and progressive elimination of pharmaceutical residues from environment are still not completely solved. Thus, the development and availability of accurate and fast commercial analyses are highly desired. The aim of this diploma thesis was the optimization and validation of multi-residue UHPLC- MS/MS analytical method designated for the determination of 52 pharmaceuticals in drinking and waste waters. The work was carried out in laboratories of ALS Czech Republic. An analytical method was subsequently used for monitoring of pharmaceuticals in both drinking and waste waters, as well as for the determination of efficiency of removing these compounds within the...

Ultrahigh pressure liquid chromatography (UHPLC) makes it possible to use very small particles (< 2 µm) as packing materials to provide high column efficiencies. Results from a careful comparison of small porous and nonporous particles show that when the particle size is small enough (< 2 µm), both porous and nonporous particles give excellent performance, and the differences in column efficiencies between porous and nonporous particles become insignificant. Columns packed with bare diamond particles could separate small molecules, especially polar molecules, however, severe tailing occurred for less polar compounds. The polybutadiene coated diamond particles gave greater retention and better separation of small molecules compared to bare particles, although no improvement in column efficiency was observed. Changes in surface bonding of thermally hydrogenated diamond particles was achieved by chemical modification using various organic peroxides with or without reagents containing long carbon chain functional groups. It appears that the alkyl groups were attached onto the diamond surface with limited coverage. LC experiments did not demonstrate good separation; however, changes in LC behavior were observed. A repetitive solvent programming approach was successfully applied to the analysis of a continuous sample stream in microbore LC. Each analysis cycle consisted of three steps: pseudo-injection, elution and rinse. In the pseudo-injection step, elution with a non- or poor-eluting solvent produced a concentrated sample plug due to on-column focusing. Factors influencing peak symmetry, resolution and analysis cycle length were investigated. Quantitative analysis of a continuous sample stream is possible under certain operating conditions. Electric field gradient focusing (EFGF) devices with distributed resistor substrates could focus proteins in the separation channel, however, the focused bands were not stable, and the repeatability was poor due to the formation of bubbles and pH gradient in the separation channel. Both fiber-based and porous glass capillary-based planar EFGF devices with changing cross-sectional area (CCSA) channels were constructed and evaluated with the aid of a home-made scanning laser-induced fluorescence detection system. The fiber-based CCSA EFGF devices gave poorer performance compared with glass capillary based devices. Porous glass capillary-based EFGF devices could focus single proteins and separate mixtures of two to three proteins.

High Pressure Liquid Chromatography (HPLC) is a well-known and widely used analytical technique which is prevalent throughout the pharmaceutical industry as a research tool. Despite its prominence HPLC possesses some disadvantages, most notably slow analysis time and large consumption of organic solvents. Ultra Pressure Liquid Chromatography (UPLC) is a relatively new technique which offers the same separation capabilities of HPLC with the added benefits of reduced run time and lower solvent consumption. One of the key developments which facilitate the new UPLC technology is sub 2-µm particles used as column packing material. These particles allow for higher operating pressures and increased flow rates while still providing strong separation. Although UPLC technology has been available since early 2000, few laboratories have embraced the new technology as an alternative to HPLC. Besides the resistance to investing in new capital, another major roadblock is converting existing HPLC methodology to UPLC without disruption. This research provides a framework for converting existing HPLC methods to UPLC. An existing HPLC method for analysis of Galantamine hydrobromide was converted to UPLC and validated according to ICH guidelines. A series of statistical evaluations on the validation data were performed to prove the equivalency between the original HPLC and the new UPLC method. This research presents this novel statistical strategy which can be applied to any two methodologies to determine parity. / Chemistry

Vid positronemissionstomografi (PET) kräver vissa radiofarmaka lösningsmedlen N,N-dimetylformamid (DMF) och dimetylsulfoxid (DMSO) vid syntes. Rester av lösningsmedlen kan därför finnas kvar i produkten som ska injiceras i patienten och därför ska mängden lösningsmedelsrester kontrolleras enligt Europeiska läkemedelsmyndighetens riktlinjer (EMA). Gaskromatografi är standardmetod för att kvantifiera lösningsmedelsrester men är inte optimal för DMF och DMSO, som istället kan analyseras med UHPLC (ultra high performanc liquid chromatography) och UV-detektor. Syftet med projektet var att utföra metodoptimering och validering av en reverse phase UHPLC-UV metod för lösningsmedlen DMF och DMSO, på ett Agilent 1290 instrument. Sex aktuella radiofarmaka (spårmolekyler) användes vid utförandet. För metodoptimering studerades flera instrumentinställningar för analys av DMF och DMSO. Under optimeringen analyserades DMF och DMSO lösta i provmatriser och spårmolekyler med olika koncentration. DMF separerade från provmatriser och spårmolekyler. DMSO co-eluerade med de nämnda ovan och därför utfördes den efterföljande valideringen endast för DMF. Resultaten från valideringen erhöll godkända resultat för fem av sex radiofarmaka. Korrelationskoefficient var 0.9998, noggrannheten bestämdes till 96.8 – 101.5% och för precision som repeterbarhet och reproducerbarhet blev den relativa standardavvikelsen &lt;5%. Detektionsgränsen bestämdes till 0.014 µg/mL och kvantifieringsgränsen till 0.047 µg/mL. Slutsatsen av projektet var att haltbestämning av lösningsmedlet DMF kan utföras. Därmed kan kraven från EMA säkerställas, men för en mer optimal metod kan ändring av pH på mobilfasen, flödeshastighet, kolonntyp och orsak till de breda topparna studeras närmare.

Topical inquiries for the Swedish Defence Research Agency (FOI) include analysis of explosive substances in different sample types. Research into explosives in complex matrixes can provide an analytical support function for forensic investigation i.e. tools for areas such as finding bomb factories, identification and risk analysis of home-made explosives (HME) and improvised explosive devices (IED) as well as preventive measures against maliciously intended use of explosives. Additionally, the research may lay the groundwork for indications of health- and environmental hazards. Utilizing state-of-the-art equipment and years of extensive expertise, FOI is able to carry out these types of research tasks to provide security and sustainability for society. The aim of this thesis project is to establish and validate developed methods for collecting, extracting, separating, and detecting trace amounts of explosives in various bodies of water using a solid-phase extraction (SPE) robot and a high-resolution (HR) mass spectrometer (MS) connected to an ultra-high-performance liquid chromatograph (UHPLC). Particular areas of interest include locations in the Stockholm archipelago where experimental detonations of explosives have taken place. Overall, UHPLC-HRMS analysis provides a powerful tool for analyzing explosives in complex matrixes with unambiguous and reliable measurement data. The compounds of investigation were hexogen (RDX), octogen (HMX), pentyl (PETN), and trotyl (TNT). To summarize, during the course of the thesis, trace amounts of some explosives were detected and quantified in various bodies of water. Furthermore, the applied method for the project was successful in qualitatively and quantitatively analyze the compounds of interest with limit of detection ranging between 0.33–11 μg/L (ppb) in various water sources.

Until now, most metabolomics protocols have been optimized towards high sample throughput and high metabolite coverage, parameters considered to be highly important for identifying influenced biological pathways and to generate as many potential biomarkers as possible. From an analytical point of view this can be troubling, as neither sample throughput nor the number of signals relates to actual quality of the detected signals/metabolites. However, a method’s selectivity for a specific signal/metabolite is often closely associated to the quality of that signal, yet this is a parameter often neglected in metabolomics. This thesis demonstrates the importance of considering selectivity when developing NMR and LC-MS metabolomics methods, and introduces a novel approach for measuring chromatographic and signal selectivity in LC-MS metabolomics. Selectivity for various sample preparations and HILIC stationary phases was compared. The choice of sample preparation affected the selectivity in both NMR and LC-MS. For the stationary phases, selectivity differences related primarily to retention differences of unwanted matrix components, e.g. inorganic salts or glycerophospholipids. Metabolites co-eluting with these matrix components often showed an incorrect quantitative signal, due to an influenced ionization efficiency and/or adduct formation. A novel approach for measuring selectivity in LC-MS metabolomics has been introduced. By dividing the intensity of each feature (a unique mass at a specific retention time) with the total intensity of the co-eluting features, a ratio representing the combined chromatographic (amount of co-elution) and signal (e.g. in-source fragmentation) selectivity is acquired. The calculated co-feature ratios have successfully been used to compare the selectivity of sample preparations and HILIC stationary phases. In conclusion, standard approaches in metabolomics research might be unwise, as each metabolomics investigation is often unique.  The methods used should be adapted for the research question at hand, primarily based on any key metabolites, as well as the type of sample to be analyzed. Increased selectivity, through proper choice of analytical methods, may reduce the risks of matrix-associated effects and thereby reduce the false positive and false negative discovery rate of any metabolomics investigation.